Eicosapentaenoic acid (EPA) induced apoptosis in human hepatoma cells throughp53 pathway.

摘要

Eicosapentaenoic acid (EPA) has been demonstrated to induce apoptosis and cell cycle arrest in various cancer lines in vitro. In this study, we investigated the anti-tumor effects of EPA on hepatoma cell lines and the potential mechanisms of induction of apoptosis.; The three hepatoma cell lines tested have different p53 status: HepG2 has wild-type (wt) p53, Hep3B has its endogenous p53 deleted, and Huh7 has a mutated p53. Tetrazolium salt, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay showed that the viability of HepG2 cells was significantly decreased after exposure to EPA, and the cytotoxicity of EPA was time- and dose-dependent. Both Fas and Fas ligand (Fas-L) mRNA expression in HepG2 cells were up-regulated after exposure to EPA. On the other hand, an increase in the Fas-L mRNA expression in Hep3B and Huh7 cells was not observed after exposure to EPA. Most notably, EPA-induced apoptosis in HepG2 cells could be inhibited almost completely by Fas-L antisense oligonucleotide.; To have a better understanding of the function of wt p53 in human hepatocellular carcinoma (HCC), we have made an attempt to establish a cell line which has inducible and double-stable expression of wt p53 with a retrovirus-mediated Tet-Off system in Hep3B cell line (of which the p53 gene is deleted).; We then utilized the established high p53 producer, Hep3B/tet/p53-13 cell line, to further explore the function of wt p53 by controlling the “on or off” status or different expression levels.; Interestingly, Hep3B/tet/p53-15 cell line was extremely different from Hep3B/tet/p53-13 cell line in its responses to the induction of wt p53. We observed that the induced levels of wt p53 could dictate the response of the cells in such a way that a higher level of wt p53 expression would result in apoptosis whereas a lower level of wt p53 would only result in cell cycle arrest without apoptosis. Additionally, induction of p53 could up-regulate the expression of p21WAF1 in Hep3B/tet/p53-13 cells and Hep3B/tet/p53-15 cells.; In the last part of this study, we attempted to gain more insight into the molecular mechanism of EPA-induced cell killing by analyzing the apoptotic pathway with the established Hep3B/tet/p53-15 cell line. Our results showed that EPA resulted in significant loss of cell viability and induction of apoptotic death in the expressing-p53 cells, as evidenced by MTT assay, acridine orange (AO) staining and DNA fragmentation analysis.; Hep3B/tet/p53 cell lines are very useful tool for research into the function of p53 and the application of p53 as a possible target for gene therapy. (Abstract shortened by UMI.)